Immortalized cell lines and methods of making the same

ABSTRACT

The present invention relates to methods of immortalizing various primary cell cultures, including pituitary cells, neurons, beta islet cells, glial cells, corneal epithelial cells and follicular stellate cells. The primary cells are transfected with a vector containing an establishment oncogene, resulting in non-transformed immortalized cells. The primary cells and/or the subsequent immortalized cells are cultured in a defined media containing one or more environmental factor(s) that control the proliferation and/or differentiation of the cells.

FIELD OF THE INVENTION

[0001] The present invention relates to methods of immortalizing primarycells and to the immortalized cell lines produced by these methods.

BACKGROUND OF THE INVENTION

[0002] The desirability of generating immortalized cell lines for use asin vitro models of various tissues has long been recognized. Forexample, researchers have endeavored for some time to generate asuitable in vitro model for cells of the anterior pituitary gland. Theanterior pituitary gland of mammals is composed of specialized cellsknown to synthesize and secrete a variety of hormones that regulatecritical body functions. Corticotropes express adrenocorticotropichormone (ACTH) which regulates the steroid hormone output from theadrenal gland. Gonadotropes express the gonadotropin hormones,luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whichoperate in concert to control reproductive functions. Thyrotropes arethe source of thyroid-stimulating hormone (TSH), which regulates thesynthesis and release of thyroid hormones from the thyroid gland.Somatotropes express growth hormone (GH), which controls cell growth.Lactotropes express a hormone closely related to growth hormone,prolactin which controls lactation and other functions as well.Prolactin was discovered in 1970 and its entire functional role is notyet understood (Hodson, Calif., 1996). Melanotropes expressmelanocyte-stimulating hormone (MSH), which regulates pigmentation ofthe skin. The suffix “troph” may be substituted for “trope” whenreferring to these cells. Control of end-organs regulated by anteriorpituitary hormones is closely regulated by circulating end-organhormones and neural inputs which interact at pituitary and hypothalamicsites to maintain homeostasis.

[0003] Some pituitary cell lines presently exist but these are usuallyneoplastic cells which continue to divide in vitro by virtue ofmalignant transformation, e.g., the GH-3 rat cell line was derived froma pituitary tumor. GH-3 cells are termed somatomammotrophs because theysynthesize and secrete both prolactin and growth hormone. Other ratpituitary hormone-secreting cell lines are also known, e.g., GH-1, whichwas derived from the same tumor as the GH-3 line. The RC-4B/C cell lineis an apparent mix of corticotropes, thyrotropes, lactotropes andsomatotropes. Likewise, with regard to human pituitary cell lines, mostof these come from neoplasms of the pituitary. Prolactinoma is the mostprevalent form of pituitary neoplasm in man. Therefore, human lactotropeadenoma cells have been extensively studied to determine the geneticmechanisms of tumor formation; such studies suggest that expression ofthe hst gene encoding fibroblast growth factor may be associated withpituitary tumorigenesis (Cai, W Y, et.al., 1994; Gonsky, R et.al.,1991). Hydridomas formed by fusion of malignant cells with gonadotropeshave been used to express human FSH (U.S. Pat. No. 4,383,034). LH hasalso been produced by fusion of human pituitary adenoma cells with ahuman lymphoblastoid cell line (U.S. Pat. No. 4,383,035).

[0004] Transformed cells are not the same as non-transformed cellspresent in the body. Transformation shifts the expression of endogenousproteins significantly, turning off the expression of some proteins,while the expression of other proteins is increased. Transformation alsoalters morphological and cellular properties, e.g. transformed cellsrequire lower amounts of serum and growth factors to supportproliferation. Transformed cells may also erroneously process proteinsresulting in molecular alteration of endogenous proteins. Humancytomegalovirus immediate-early promoter was more efficient in directingreporter gene expression when expressed in transformed thannon-transformed rat anterior pituitary cells suggesting thattransformation alters transcriptional factors (Coleman, T A, et.al.,1991). Thus, a transformed pituitary cell may not reflect the normalphysiological and cellular processes of its progeny.

[0005] Hormone-secreting cells of the pituitary can be grown in vitro asa primary culture. Such primary cultures of pituitary cells have beenused extensively to investigate physiological processes involved inregulation of hormone secretion. For example, these cultures were usedto bioassay specific hypothalamic releasing hormones that regulatehormone output of the pituitary. This discovery provided considerableinsights into the mechanisms of intercommunications between the nervousand endocrine systems (Guillemin, 1978). However, after several celldivisions primary cultures invariably reach a crisis stage andthereafter cease to divide. This property is referred to as senescence.There are some pituitary cell lines which continue to proliferate inculture, but are apparently non-transformed. Mouse thyrotrope tumorssuch as TtT 97 result from thyroidectomy, synthesize and release TSH,and respond to T3 and TRH (Furth, J, 1955; Condliff, P G, et.al., 1969;Cacicedo, L, et.al., 1981). The rat somatomammotroph cell line, rPCO andclonal derivatives, was derived from primary culture of rat pituitarycells in media containing T3 and GHRH, which may have resulted inselective proliferation of somatotropes. RPCO cells synthesize andrelease growth hormone and prolactin; secretion is differentiallyaffected by TRH, T3 and GHRH (Chomczynski, P, et.al., 1988; Kashio, Y,et.al., 1990). Both of these cell lines have been used in basic researchinto the molecular biology of pituitary hormone processing (Coleman, TA, et.al., 1991; Wood, W M, et.al., 1989). The mechanisms by which thesecells are immortalized is unknown. These cells have not yet beendefinitively demonstrated to be non-transformed (Chomczynski, P, et.al.,1988).

[0006] Recombinant DNA technology has also been used to produce proteinhormones of the pituitary gland. The non-glycosylated monomers of growthhormone and prolactin were initially expressed in E. coli cells using arecombinant plasmid containing the cDNA for growth hormone or prolactin.Recombinant protein accumulates in inclusion bodies primarily as reducedmonomers. Following solubilization with urea, reoxidized monomers wererecovered through use of reoxidation procedures which resulted inbiologically active material similar to native hormones with theaddition of a methionine residue at the amino terminus (Paris, N,et.al., 1990). The group of heterodimeric glycoprotein hormonesincluding hCG, LH, FSH and TSH have also been expressed in ChineseHamster Ovarian (CHO) cells using cloned sequences of both thehomologous α-subunit and the hormone-specific β-subunit (Reddy, V B,et.al., 1985; Simon, J A, et.al., 1988; Keen, J L, et.al., 1989; U.S.Pat. Nos. 4,923,805; 5,156,957; 4,840,896). These heterodimeric proteinsare known to be glycoproteins containing 15%-35% carbohydrate asN-linked and O-linked glycans present on both the α and β-subunits.These carbohydrates add structural complexity and have various functionsin the assembly, stabilization, modulation of biological activity andcontrol of clearance of these molecules (Szkudkinski, M W, et.al., 1996;Galway, A B, et.al., 1990). The addition of carbohydrate moieties to theprotein backbone is a function of the host cell and since these aredifferent from the natural hormone-producing cells, variation in theglycosylation of recombinant hormones may result. Detailed comparison ofurinary and recombinant human FSH (rhFSH) has shown that rhFSH is moreacidic, suggesting differences in sialic acid content or terminalmonosaccharides (de Leeuw, R, et.al., 1996). Also, recombinant human TSH(rhTSH) contains only sialic acid at terminal biantennarymonosaccharides while pituitary-derived hTSH also contains sulfatedN-acetyl galactosamine terminal residues. This results from the lack ofN-acetyl galactosamine transferase in CHO cells used for the expressionof rhTSH (Szkudlinski, M W, et.al., 1996).

[0007] There are non-transformed cells in the art which are thought toarise through spontaneous immortalization as occur in non-malignant orbenign tumors. However, these cell lines are relatively rare. Some othercells are known to undergo spontaneous immortalization withouttransformation. Studies of such cells showed that c-myc was overexpressed while expression of other establishment oncogenes wasunaltered, possibly due to a chromosomal translocation (Tavassoli &Shall, 1988; Madsen, et.al., 1992). Transfection of the v-myc oncogeneinto a murine macrophage cell line resulted in immortalization,suggesting that over expression of the myc oncogene itself wassufficient to induce immortality. Furthermore, the biological propertiesof the immortalized cell line were similar to normal cells (Blasi, etal., 1987). A variety of other studies have also demonstrated theimmortalization effect of v-myc transfection into several differenttypes of cells, including those with secretory functions common toendocrine cells (Strom, et.al., 1991; Bernard, et.al., 1989; Briers,et.al., 1993; Vanderstichele, et.al., 1994; Briers, et.al., 1994;Hoeben, et.al., 1995).

[0008] Expression of the cellular myc gene is normally repressed byLef/Tcf transcription factors which bind to specific sequences of thec-myc promoter (He, TC, et.al., 1998). In some forms of cancer andnormal development, c-myc expression is increased through disruption ofthe developmental pathway known in the art as Wnt. The adenomatouspolyposis coli gene (APC) is a tumor suppressor gene that is mutated inabout 85% of all human colon cancers. The APC gene product is acomponent of the Wnt pathway that normally complexes β-catenin,preventing its effect on the nucleus. Mutations of the APC gene oractivation of the Wnt pathway via ligand-receptor interaction result indisruption of the APC-β-catenin complex allowing β-catenin access to thenucleus. β-catenin then binds to Tcf transcription factors, de-repressesthe c-myc gene resulting in its increased transcription (He, TC, et.al.,1998; Dale, 1998). Increased cell proliferation in response todevelopmental signals and in colon cancer is mediated by increased c-mycexpression.

[0009] The protein product of the myc gene is a transcription factorwhich forms a heterodimeric complex with another protein, Max. Myc is acentral regulator of cell proliferation through transcription effects,including repression and activation of target gene expression, e.g.,MrDd and cdc25A (George, K H; 1996). The latter target also inducesapoptosis in cells. Max also forms homodimers and heterodimers with Madand Mxi-1, alternative partners to Myc, which compete with Myc/Max forcommon gene targets. These various interactions are thought tb regulatethe ultimate effect of Myc over expression, i.e., cell differentiation,immortalization without transformation, transformation, or cell death(Facchini, L M and Penn, L Z; 1998; Desbarats, L, et.al., 1996; Amati, Band Land, H; 1994).

[0010] The SV40 virus has been used to immortalize several cells ofanimal and human origin. The pSV3neo plasmid containing the completeSV40 early genetic region including the large T and small T antigens wasused to immortalize rat Leydig cells (Nagpal, M L, et.al., 1994). Twocell lines resulted which were maintained for 35 passages withoutapparent transformation as indicated by an absence of theanchorage-independent growth in soft agar. The large T antigen codingsequence had become integrated into the cellular genome. These cellsdisplayed many characteristics of differentiated Leydig cells includingexpression of LH receptors (LH-R), insulin-like growth factor I (IGF-I)and IGF-I receptors (IGF-IR) and IGF binding protein 2 (IGFBP-2). Theamounts of transcripts of the LH-R gene were lower, IGF-I, IGF-IR werethe same and IGFBP-2 were much higher in the immortalized cells. Also,the immortalized cells could not synthesize testosterone due to lowlevels of the enzyme P450 scc. Hence, the immortalized cells maintainedsome, but not all, differentiated characteristics of rat Leydig cells(Nagpal, M L, et.al., 1994). In order to avoid production of virusparticles in permissive and semipermissive cells, including human andmonkey cells, SV40 plasmids containing the large T antigen gene weremodified to block viral replication by deleting part of the viral originof replication, the so-called ori⁻ SV40 mutant (Gluzman, 1980). Suchmutants have been particularly valuable in the immortalization of humancells (Chow, J Y, 1989), including granulosa cells, fetal liverepithelial cells, breast and other epithelial cells (Byong-Lyul, L,et.al., 1996; Ishida, T, et.al., 1995; Berthon, P, et.al., 1992;Lechner, M S and Laimins, L A, 1991). Human cell lines immortalized bySV40 Large T antigen exhibit increased growth in culture. However, theyusually reach secondary senescence and are no longer viable (Stein, G H,1985).

[0011] The degree of differentiation of the cells depends on the statusof differentiation at the time of transfection. Thus, fetal liver cellsimmortalized by the plasmid pMK16-SV40(ori⁻) failed to expressa-fetoprotein (AFP) possibly because it had not yet differentiated atthe time of establishment of the cell line (Ishida, T, et.al., 1995).The SV40 large T antigen is thought to combine with the products of thetumor suppressor genes p53 and p105-Rb, neutralize anti-oncogeniceffects of these genes, including activation of apoptosis and therebyincrease cell proliferation. Such effects may also lead to malignanttransformation. Two immortalized breast epithelial cell lines resultedfrom transfection by the SV40 large T antigen. One of these linesremained non-transformed while another resulted in a transformed cellline, showing that SV40 large T antigen can result in immortalizationand malignant transformation (Berthon, P, et.al., 1992).

[0012] The temperature sensitive mutant of SV40 large T (tsA58) has alsobeen used to immortalize cells, including human pituitary thyrotropes.At the permissive temperature, the large T antigen is expressed andcells exhibit a transformed phenotype. At the non-permissivetemperature, large T antigen is no longer expressed and cells revert tothe normal differentiated phenotype (Chou, J Y, 1989). Adult humanthyrotropes transfected with tsA58 plasmid proliferated at thepermissive temperature 33° C. and showed no growth at 39° C. These cellshave undergone more than 150 passages. Contrary to adult thyrotropes,these immortalized thyrotropes did not express TRH receptors nor dothese cells secrete hTSH (Ham, et.al., 1998). The large T antigen ofSV40 has also been used to immortalize specific pituitary tumor cells atdiscrete stages of development by constructing large Tantigen-containing transgenes driven by various promoters that aredifferentially activated during development (Windle, J J, et.al., 1990;Alarid, E T, et.al., 1996). A hybrid of the adenovirus-12 and the SV40virus has also been used to immortalize human prostate cells. Thesecells express many of the normal phenotypic characteristics of humanprostate cells and were non-tumorigenic (U.S. Pat. No. 5,610,043).

[0013] Accordingly, a need exists for methods of immortalizing primarycultures of various cell lines that are not transformed. The presentinvention satisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

[0014] The present invention relates to novel methods for the generationof immortalized cell lines from primary cultured cells and to suchimmortalized cell lines. Such cell lines are immortalized lines derived,for example, from hormone-producing cells of the pituitary gland, i.e.,lactotropes, somatotropes, thyrotropes, gonadotropes, corticotropes ormelanotropes. Supporting cells normally associated with endocrine cellssuch as follicular stellate cells may also be immortalized as describedherein as well as neurons, glial cells, corneal epithelial cells, andγ-islet cells of the pancreas which produce insulin.

[0015] Such cell lines are immortalized but not transformed as occursduring malignant transformation of a normal cell into a cancerous cell.Hence the immortalization of these cells is not secondary to malignanttransformation. Furthermore, the cells of a given line are maintained inculture under conditions allowing expression of the adult phenotype ofthe cell. For pituitary cells, the adult phenotype includes propertiessuch as: presence of the appropriate hormone and secretoryultrastructure, secretion of such hormone in response to thosesecretagogues which normally regulate hormone secretion in the adultorganism, receptors for the hypothalamic releasing hormone normallypresent on adult cells, expression of hormone-encoding genes, andexpression of appropriate differentiation factors such as, for example,Pit-1 or SF-1 (Simmons, D M, et.al., 1990; Sanno, N, et.al., 1998;Bedford, et.al., 1996).

[0016] The present invention also refers to immortalization of cellsfrom different species. Thus primary cells immortalized according to thepresent invention may be derived from various species, including forexample, human, equine, bovine, canine, rat or mouse.

[0017] A feature of the present invention is that it involves use ofboth environmental factors and genetic manipulation of primary culturedcells to achieve or maintain immortalization without transformation atthe fully differentiated phenotype. Environmental factors, such as theosmolarity of the cell culture media, addition of various growth factorsand tissue extracts have been used to extend the longevity of culturedpituitary cells (e.g., U.S. Pat. Nos. 4,124,448 & 5,747,341).Significant effects of environmental factors on primary culture ofpituitary cells, including effects on differentiation and proliferationhave also been observed. In one embodiment, the present inventioninvolves use of environmental factors to induce specific states ofdifferentiation and/or proliferation prior to use of establishmentoncogenes including, for example, the large T antigen of the SV40 virusand controlled expression of the myc proto-oncogene to immortalizecells. In another embodiment, specific environmental conditions can beused to maintain immortalized cell cultures that already possess desiredproperties such as a particular state of differentiation, hormoneexpression or proliferation without the need to induce such states.Furthermore, environmental conditions may also be used to control theexpression and differentiation of cells that have been immortalizedaccording to the methods of the present invention.

[0018] The present invention offers significant advantages over theprior art. This invention provides an alternative source of relativelypure native protein hormones for various applications in research,diagnostics and therapeutics. Presently, native material is derivedthrough the extraction of tissues and the purification of each hormonepresent within such tissue. The physical and chemical properties of somehormones are closely related, for example pituitary growth hormone andprolactin are structurally similar. Prolactin is thought to have evolvedfrom growth hormone (Cooke, N E, et.al., 1981). Thus, completeelimination of one hormone from the other is difficult; usually,purified hormone is at least partially contaminated with the other.

[0019] The present invention provides for cultures of purehormone-producing cells without contamination by cells which produceother hormones. Thus, for example, this invention allows a pure cultureof immortalized lactotropes without somatotropes. Likewise a clonalculture of pure somatotropes is possible without the presence oflactotropes. Hence, cultures of immortalized lactotropes provide a crudeprolactin sample devoid of contamination by growth hormone andsomatotrope cultures provide crude growth hormone uncontaminated byprolactin. Optimization of the secretion of the desired pituitaryhormone provides additional advantages in purification to homogeneity asthe hormone content in the culture media may be enriched, while othercontaminating proteins are minimized. Purification methods well-known inthe art (Sinha, Y N) may then be applied to culture media enriched bythe secretion of, for example, prolactin or growth hormone exclusivelyto generate highly purified material uncontaminated by related hormones.Absolute purity of hormones is desirable in such applications as hormonestructure-function determinations and therapeutic uses of thesehormones. Gonadotropes are known to produce both LH and FSH. Hence,gonadotrope cultures do not provide sources of impure hormone devoid ofgonadotropin contamination.

[0020] Another advantage of the present invention in its use as analternative source for native pituitary hormones concerns the state ofglycosylation of the heterodimeric glycoprotein hormones LH, FSH andTSH. Glycans are added post-translationally to the polypeptide chainscomposing the alpha and beta subunits. Glycosylation is a property ofthe cell expressing the hormone. As previously noted, rhTSH containsonly sialic acid at terminal biantennary monosaccharides whilepituitary-derived hTSH also contains sulfated N-acetyl galactosamineterminal residues resulting from the lack of N-acetyl galactosaminetransferase in CHO cells used for the expression of rhTSH (Szkudlinski,M W. et.al., 1996). Since the present invention utilizes the endogenouscell from which native hormone arises, such a source can provideglycosylation patterns closer to native hormones than heterologouscells. The structure of the carbohydrate containing moieties containedin pituitary hormones is known to be an important determinant offunction. A study comparing pituitary-derived human FSH, rhFSH,chemically deglycosylated FSH, and FSH expressed in a baculovirusexpression vector/Sf9 cell system which fails to glycosylate FSH foundsubstantial loss of biological activity in the deglycosylated forms ofFSH. FSH deglycosylation is known to result in inhibitory intracellularevents (Arey, et.al., 1997). Thus while the complete structure-functionrelations of FSH are not yet known, glycosylation is a key structuraldeterminant of function. Hence, use of the present invention to producelarge quantities of native glycoprotein hormones is likely to advancedetailed studies of structure and function of these hormones. Thepresent invention also provides another source of natural isoforms ofnative protein hormones for detailed study of their functional roles.Another advantage of the present invention is that cell lines or donorsmay be tested for viral contamination, including; HIV 1 & 2, HBV, HCV,etc prior to immortalizing the cells, thus providing a safe source ofnative pituitary hormones.

[0021] Additionally, the present invention allows investigation of themolecular biology of pituitary hormone producing cells in vitro withoutcompromise of results due to cellular transformation. For example,determination of the molecular biology of the transforming growth factorfamily of proteins including activin, follistatin and inhibin is anappropriate application of the immortalized gonadotropes and othertarget cells. Many of the interactions between regulatory pathways maybe altered by transformation. Since the cells of the present inventionare not transformed, natural regulatory pathways are intact and suitablefor direct investigation. The molecular biology of particular cells maybe utilized to optimize expression of a given pituitary hormone and itssecretion. Manipulation of these latter parameters effects the specificcellular expression of a given pituitary hormone. Higher levels ofspecific cellular expression are desirable methods to reduce the cost ofthe production of a therapeutic product such as cell line-derived FSH.Furthermore, cellular molecular biology may be utilized to extend thefunctional lifetime in culture, also resulting in a lower productioncosts.

[0022] A final advantage of the present invention concerns the abilityto immortalize human pituitary hormone producing cells. While transgenicapproaches have been used to immortalize rat pituitary cells at variousstages of differentiation, such studies are not realistically applicableto humans because of ethical and regulatory issues regarding transgenichuman beings. However, since the present invention utilizes primarycultured cells, including human pituitary cells, such cells may beimmortalized and various embodiments of the present invention can beapplied to immortalization at different stages of differentiation, giventhat these can be established in primary culture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a graph of the growth of primary cultures of pannedhuman pituitary cells. The total number of cells determined byhemacytometer is shown as a function of time in different culture media.Cultures indicated by ▪ were grown in RPMI-based Gc medium. The □represent cultures grown in medium 199-based Gc medium. The growth ofcultures in Opti-MEM alone x or Opti-MEM plus glucose and nonessentialamino acids ▴ is also shown.

[0024]FIG. 2 shows the effects of different media on growth hormonesecretion from panned human pituitary cells. Growth hormone secretion inng/ml is shown as a function of days in culture for first passage cells.The effects of RPMI-based Gc medium with estradiol on monolayer cultures▪ or spheroid cultures ♦ is compared to DMEM/F12-based Gc media with □or without Δ estradiol.

[0025]FIG. 3 shows the optimization of liposome-based transfection ofbovine pituitary cells using Lipofectin®. Bovine pituitary cells plated24 hours prior to transfection with reporter plasmid, pcDNA3.1/His/LacZ(InVitrogen, San Diego, Calif.). The efficiency of transfection is shownat various DNA to lipid ratios in μg/μl (ug/ul) when the cells wereplated at 50,000 cells/ml (open bars) or 30,000 cells/ml (solid bars).

[0026]FIG. 4 depicts plasmids in which the myc gene is shown inserted inthe pLXSN into the multi-cloning site yielding an expression vector inwhich the promoter in the 5′ LTR drives transcription of the myconcogene (A). The myc oncogene inserted in the pLXCX2 vector downstreamfrom the constitutive promoter, CMV is shown in (B), while (C)illustrates the myc oncogene inserted into pLXSN where the SV40 promoterelement is replaced with an inducible promoter (P) such as aglucocorticoid response element or the tetracycline response element.These plasmids can replicate in bacteria and mammalian cells as well. ψis the retroviral recognition site for packaging of these plasmids in ahelper strain of cells which provides the viral coat proteins.

[0027]FIG. 5 shows the growth of different cell lines in athymic mice.(A) shows the results from the positive control, MCF-7 cells. Theresults obtained from immortalized bovine cell lines, C1+(B) and C3-1(C) are also illustrated. The volume of tumor growth is shown as afunction of days post implant. The Y axis scale differs in (A), (B) and(C). Different symbols are from five different mice injected with 10million cells subcutaneously.

[0028]FIG. 6 shows RT-PCR of α-subunit, growth hormone, and prolactinRNA. Products of RT-PCR were analyzed on a 2% NuSieve/1% agarose gel runat 100V in 1×TAE buffer (40 mM Tris-acetate and 1 mM EDTA). (A)α-subunit product, lane 1; α-subunit product digested with XbaI, lane 2;HaeIII, øX174 marker DNA, lane 3; α-subunit product, lane 4; α-subunitproduct digested with HinfI, lane 5; growth hormone product, lane 6;growth hormone product digested with BanII, lane 7; prolactin product,lane8; prolactin product digested with PvuII, lane 9. (B) LTA5,α-subunit product, lane 1; LTA5 α-subunit product digested with XbaI,lane 2; LTA5 α-subunit product digested with HinfI, lane 3; HaeIII,øX174 marker DNA, lane 4; LTA6, α-subunit product, lane 5; LTA6α-subunit product digested with XbaI, lane 6; LTA6 α-subunit productdigested with HinfI, lane 7; RT-PCR reaction containing RNA for LTA5,LTA6, and LTA8 without added reverse transcriptase, lane 8; RT-PCRreaction using the cell line LnCap, lane 9; α-subunit product fromprimary pituitary cells, lane 10; HaeIII, øX174 marker DNA, lane 11;LTA8, α-subunit product, lane 12; LTA6 α-subunit product digested withXbaI, lane 13; LTA6 α-subunit product digested with HinfI, lane 14.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The methods of the present invention can be performed accordingto the following steps: 1. Primary culture of dispersed adult or fetalcells. 2. Expansion of the primary cultured cells under definedenvironmental conditions (also referred to as “environmental factors”herein) including the physical substrate to which attachment-dependentcells attach and the liquid culture medium exposed to said cells. 3.Transfection of primary cultured cells by transfection methods usingplasmid DNA containing a DNA vector designed to ensure expression of aforeign gene that induces immortalization including, but not limited to,an establishment oncogene such as viral oncogenes, cellularproto-oncogenes, tumor suppressor gene regulators, or their derivatives.4. Selection of transfected cells. 5. Characterization of the selectgenotypic and phenotypic traits of cells which proliferate beyondsenescence of wild-type cells maintained in identical conditions.Wild-type cells refer to cells that have not been transfected byplasmids containing foreign DNA establishment oncogenes or theirderivatives. The following sets forth the detailed description of themethod steps of the present invention.

[0030] 1. Primary Culture of Dispersed Cells.

[0031] Primary culture is the means by which cells are established incell culture according to methods known in the art. For example, apituitary specimen is collected for primary culture using methods tomaximize viability of pituitary cells. Animal pituitary glands may beobtained from a slaughter house at the time of extraction and stored instandard cell culture media, e.g., DMEM/F12. Fetal human pituitaryglands are procured according to NIH guidelines for use of human fetaltissue and stored in cell culture media following extraction. The timefrom extraction to dispersion is less than 24 hours for human pituitarytissue and within 6 hours of extraction for bovine pituitary tissue.

[0032] Tissue is dispersed by a combination of mechanical and enzymaticprocedures well known in the art. For example, mechanical dispersionoccurs first by mincing human fetal tissue with surgical scalpels or bysurgical dissection of adult bovine pituitary glands followed by sievingthrough a small pore screen. Mechanically dispersed human pituitarytissue is then incubated in 1 mg/mL collagenase in culture medium at 37°C. for 45 minutes; dispersed bovine pituitary tissue is treated with0.2% trypsin, 1 mg/mL hyaluronidase and 1 mg/mL collagenase inphysiological saline for 60 minutes at 37° C. Many variations of thesedispersion methods are possible and are readily determined by thoseskilled in the art. Following enzymatic treatment, enzymes are washedfrom the tissue by use of physiological saline or culture media andseparation of the tissue by centrifugation. This wash procedure isrepeated as needed to complete enzyme washout.

[0033] 2. Expansion of Primary Cultured Cells.

[0034] The dispersed cells are then resuspended in media used forprimary culture. Those skilled in the art can readily determine theappropriate media depending on the species from which the cells arederived. For bovine pituitary cells, this media can be, for example,minimum essential media (MEM), 10% Fetal Bovine Serum (FBS), 1%penicillin/streptomycin and 1% mycostatin. For culture of humanpituitary cells, useful culture media include, without limitation: a)Serum-free media such as Gc (Hedlund and Miller, 1994) supplemented withcommonly used factors well-known in the art. Bovine pituitary cells areplated directly onto standard styrene tissue culture plates.

[0035] However, maintenance of hormone secretion from cultured humancells, particularly human pituitary cells, often requires use ofextracellular matrix proteins. Purified fetuin is a well-known basementmembrane protein that allows maintenance of a well-defined medium.Cruder materials such as matrigel basement membrane matrix is a solubleprotein preparation from the Englebreth-Hoth-Swarm mouse sarcoma, atumor rich in extracellular matrix proteins. Alternatively, human tumorcells elaborating an extracellular matrix, e.g., MCF-7 cells, derivedfrom pleural effusion in 1973 (Soule, et.al., 1973) are initiallyplated, grown to confluency, ruptured by hypo-osmotic shock and used asan extracellular matrix upon which human pituitary cells are grown.

[0036] Growth factor additions to the basal media may include, but arenot limited to, activin, tri-iodothyronine, galinin, nerve growthfactor, leukemia inhibitory factor, hepatocyte growth factor, acidic orbasic fibroblast growth factor, platelet-derived growth factor,pituitary adenylate cyclase-activating polypeptide, vasopressin,retinoic acid, vasoactive intestinal polypeptide. However, it isimportant to avoid fetal bovine serum, since it suppresses hormonesecretion in primary cultured human fetal pituitary cells possibly byinducing terminal differentiation. Growth factor additions are made atany time during the expansion of these cultures to control proliferationand differentiation. The amount or concentration of growth factors,times of exposure, use of multiple growth factors and other conditionscan readily by determined by those skilled in the art and depend, forexample, on such factors as cell type, species from which the cells arederived, age of the culture and stage of differentiation. The status ofdifferentiation may be determined by a variety of well-known methods inthe art such as measurement of the levels of a differentiation-specificprotein, e.g, cytokeratin (O'Guin, et.al., 1990).

[0037] 3. Transfection of Primary Cultured Cells.

[0038] Primary cultured cells are known to be refractory to mosttransfection systems (Uyttersprot, N, et al., 1998). Therefore,transfection efficiency in cultured pituitary cells was optimized usinga plasmid (pcDNA3) containing the β-galactosidase gene and detection ofthis reporter with X-gal staining method. Liposomes containing cationiclipids (Lipofectin®) were found to be effective in transfecting culturedpituitary cells. FIG. 3 shows the results of optimization of theDNA/lipid ratios at two different plating densities. These data showthat 1 to 2 μg DNA per 4 to 6 μl Lipofectin® result in transfectionefficiencies slightly greater than 1% when the cell are plated at 3×10⁴cells/ml. Hence, liposome-based transfection using cationic liposomessuch as Lipofectin® is useful for transfecting primary cultured cells.Preferably, transfection efficiency is at least 1%, and more preferablyat least 10%. Transfer of genes into primary cells by retroviruses isknown to be highly efficient, with transduction efficiencies approaching100% (Coffin, J M and Varmus, H E, 1996; Ausubel, F M, et.al., 1994).Use of retroviral transfection methods is also useful in thetransfection of primary cultured cells according to the presentinvention.

[0039] The concept of targeting liposomal delivery to specific cells bycoupling antibodies which recognize unique cell surface antigens iswell-known (reviewed by T D Heath and F J Martin, 1986). Efficientcoupling of antibody to the liposome requires formation of covalentbonds. Methods employing heterobifunctional cross-linking reagents andcovalent linkage to polyethylene-glycol (PEG) moieties of PEG-stabilizedliposomes are well-known covalent methods that efficiently formimmunoliposomes (Hansen, C B, et.al., 1995; Shwendener, R A, et.al.,1990). The present invention also encompasses plasmid DNA-containingliposomes covalently coupled to antibodies, that are specific to surfaceantigens of pituitary and other cells, as a method of targetedtransfection of primary cultured cells. Such cell surface antigens are,for example, receptor molecules for hypothalamic releasing hormonesincluding, among others, corticotropin releasing hormone (CRH) receptorand the gonadotropin releasing hormone (GnRH) receptor. Thedistinguishing characteristic of such immunoliposome target antigen isthat it is uniquely present upon the surface of a particular target celland not similarly present on other cells.

[0040] The targeted transfection method of transferring plasmid DNA intocultured pituitary cells is applied to mixed cultures of various cellspresent in the initial cell dispersion and surviving in primary culture.Such mixed cultures are known in the art to maintain intercellularinteractions and paracrine effects of endogenous growth promotingfactors released from a particular type of cell and acting on othercells. However, another embodiment of the present invention involves useof immobilized antibody to enrich cultures of specific hormone-producingcells. Accordingly, antibodies to specific cell surface proteinsincluding pituitary hormones and receptors for hypothalamic releasinghormones are coupled for example, to magnetic beads, mixed withdispersed cells and separated by magnetic concentration. Theantibody-bead conjugate is then detached from cells by incubation inbovine serum albumin (Flaws, J A and Suter, D E, 1993; Valenti, S,et.al., 1995). Separation of cells by cell sorting is also apossibility. The separated cells are then cultured and transfected asdescribed above.

[0041] The present invention includes the use of suitable plasmid DNAthat is transferred into target cells by transfection methods outlinedabove to achieve immortalization of cultured pituitary cells. PlasmidDNA is composed of a vector and a foreign gene or genes. The vector isdesigned to support the expression of foreign DNA and its integrationinto to the genome of transfectants. The vector used to supportexpression of the foreign DNA is composed of various DNA sequenceswell-known in the current art of molecular biology. For example, asuitable expression vector is pcDNA3 (InVitrogen, Inc., San Diego,Calif.) which contains the enhancer-promoter sequences of the immediateearly gene of human cytomegalovirus (CMV) upstream from varioussubcloning sites, a neomycin resistance gene and prokaryotic sequencespermitting growth and selection in microorganisms. Other eukaroyticvectors capable of stable transfection, preferably containing the SV40,CMV, thymidine kinase (TK) or Rous sarcoma virus (RSV) promoterimmediately upstream from the subcloning site, are within the scope ofthe present invention. These vectors include, without limitation, knownretroviral expression vectors such as pLNCX2 and pLXSN (See FIG. 4).Also, the use of endogenous promoter sequences specific to normalpituitary cell expression such as the a subunit promoter engineered todrive the expression of the SV40 Large T antigen (Mellon, Pl, et.al.,1991), may also be used to transfect primary cultured pituitary cells.The foreign gene to be expressed in transfectants is subcloned intosuitable subcloning sites of the expression vector using well-knownmethods. As an aid in subcloning into the expression vector, primersused for polymerase chain reaction (PCR) of specific target genes aresynthesized to include Testriction endonucleases near the 5′ endsaccording to the subcloning sites available on the expression vector.

[0042] Expression vectors containing a variety of subcloned foreigngenes are encompassed in the present invention; the result of expressionof these genes within transfectants is controlled proliferation oftransfected cells without ensuing malignant transformation. These genesinclude, but are not limited to, known establishment oncogenes: (a)viral oncogenes, including, for example, the large T antigen of SV40,the Epstein-Barr virus and the E7 gene of the human papilloma virus, (b)cellular proto-oncogenes including, for example, gsp, gip2 (Barlier,A.,et al., 1997), myc and fos, pituitary tumor-transforming gene (Zhang,X, et.al., 1999) and c) tumor suppressor genes, including, for example,MEN1 , MEN2a and MEN2b (Chakrabarti, R, et al., 1998), p53 and p105-Rb.Derivatives, including those resulting from mutation or deletion ofspecific sequences of the above listed genes are also encompassedherein. For example, pituitary cells derived from animal species can betransfected with the SV40 large T antigen, while human pituitary cellsare more preferably transfected with expression vectors encodingsequences of c-myc, v-myc, N-myc, L-myc or derivatives thereof. Inherentin this embodiment of the present invention is regulated over expressionof myc allowing for controlled cell division without apoptosis, asopposed to unregulated myc over expression as occurs in oncogenictransformation.

[0043] One of four methods described below can be used for regulatedexpression of foreign myc transcripts within transfectants. The firstmethod involves the use of specific promoters resulting in regulatedexpression, including for example, SV40, CMV, TK or RSV. The secondmethod involves inducible myc expression by use of expression vectorpromoters, e.g., β-galactosidase, T7, chloramphenicol, mousemetallothionein (MT) promoter (Kelly, et.al., 1997), the glucocorticoidpromoter, and tetracycline response elements. The construction of aninducible promoter is shown in FIG. 4C. Inducible promoters areactivated by small, non-toxic molecules such as, but not limited to,isopropyl-β-D-thioglactopyranoside (IPTG), added to culture media (Wang,Q, et.al., 1992; Films, et.al., 1992) or Zn²⁺. The presence of aninducer drives the expression of myc inducing immortalization ofpituitary cells. When the inducer is removed from cultures theimmortalized cells revert to their differentiated state having all thecontrol mechanisms and machinery for production of hormones. An exampleof this negative control of differentiation is seen in immortalizedhuman dorsal root ganglia cells where the cells return to their neuronalphenotype when the inducing molecule for oncogene expression is removedfrom the culture (Raymon, H K, et.al., 1999). The third method involvesthe controlled expression of endogenous c-myc, as for example, by use ofcomponents of the Wnt pathway, including Wnt receptor agonists. Finally,the fourth method involves the genetic alteration of the foreign myccDNA to alter its biological activity, including but not limited to,site-specific mutagenesis resulting in Ser⁶² substitution whichselectively inhibits the transforming activity of myc (Pulverer, B J,et.al., 1994).

[0044] Another embodiment of the present invention is the promotion ofcell proliferation by environmental factors such as exposure toproliferation-inducing growth factors, for example, activin which is aspecific growth factor for gonadotropes (Katayama, T, et.al., 1990),tri-iodothyronine, galinin, nerve growth factor, leukemiainhibitory factor, hepatocyte growth factor, acidic or basic fibroblastgrowth factor, platelet-derived growth factor, pituitary adenylatecyclase-activating polypeptide, vasopressin, retinoic acid, vasoactiveintestinal polypeptide. Environmental factors also include geneticmanipulations outlined above, together with genetic blockage ofapoptosis. For example, over expression of the Bcl2 gene is known toblock apoptosis (Adams, J M and Cory, S, 1998). Hence, transfection withplasmids containing the cDNA for Bcl2 resulting in its over expressionis an alternative means of immortalization of proliferating pituitarycells.

[0045] Subsequent to immortalization of pituitary hormone-producingcells, genetic alterations resulting in enhanced hormone expression,altered hormone glycosylation, and variation of the hormone amino acidsequence may be desirable depending upon the particular application ofthe present invention. Hence, the present invention also includesgenetic alterations of a specific immortalized pituitary cell line,including, but not limited to: those alterations resulting in enhancedexpression of endogenous hormone(s), altered amino acid sequence of theexpressed hormone, and altered glycosylation of the pituitaryglycoprotein hormones.

[0046] 5. Selection and Expansion of Stable Transfectants.

[0047] Selection of transfectants can be accomplished by the survival ofimmortalized cells beyond senescence, or by the transfection of anantibiotic resistance gene within the plasmid used to induceimmortalization. For example, inclusion of the neomycin-resistance genewithin the expression vector allows for selection of transfectants bygrowth in media containing toxic levels of geneticin (G418).Furthermore, continued expansion of transfectants in selective media isa well-known method in the art for selective expansion ofplasmid-containing cells and maintaining stable transfection. Usingtargeted transfection to specific types of hormone-producing cells, forexample, further enhances selectivity in that clonal cultures ofhormone-producing cells may be established. In applications of thepresent invention on mixed cultures without targeted transfectionmethods, clonal selection of pure hormone-producing cell types, e.g.,somatotropes, may be accomplished by cloning methods well-known in theart such as use of a cloning ring or limiting dilution. Furthermore, useof defined cell culture media (i.e., the use of environmental factors asdescribed above) is preferable during expansion of transfectants tomaintain controlled states of proliferation and differentiation.

[0048] 6. Characterization of Transfected Cells.

[0049] Methods well-known in the art of cell culture are used to monitorand characterize transfected pituitary cells. Cellular viability ismeasured by standard methods of doubling time determination. Passagenumber is also determined. Survival beyond senescence of wild-type cellsis a standard indicator of immortalization. However, secondarysenescence or appearance of crisis stage following survival beyondsenescence is common in cells transfected with establishment oncogenes.Immortality generally refers to cell lines which have substantiallyoutlived senescence, both primary and secondary. As used herein,immortality refers to continued maintenance of cell viability at a5-fold greater passage number than senescence with maintenance of steadydoubling times throughout this growth period. For example, expression ofthe protein/peptide pituitary hormones is an important phenotypic traitthat the present invention is designed to maintain. Hormone expressionis commonly measured by assaying hormone secretion into culture media.This requires the use of quantitative, highly sensitive assays that havebeen rigorously validated. Such assays are not readily availablecommercially for animal hormones but several such assay systems areavailable for human pituitary hormones because of the clinicalsignificance of these measurements. Alternatively, expression ofhormones in immortalized cells can be determined by measurements ofmessenger RNA encoding specific hormones as by reverse transcriptase PCR(RT-PCR) or Northern blot analysis. Similarly, well-known methods areused to measure receptors for hypothalamic releasing hormones andexpression of differentiation factors of known importance in thedifferentiation of pituitary hormone-producing cells such as Pit1 andSF-1 (Simmons, D M, et.al., 1990; Sanno, N, et.al., 1998; Bedford,et.al., 1996). Such measurements are important indicators of the statusof differentiation of cultured pituitary hormone-containing cells.Analysis of additional markers of differentiation including cytokeratinsalso provides information on the developmental status of theimmortalized cells (O'Guin, et.al., 1990). Transformation is assessed bystandard methods well-known in the art including in vitro assays such asgrowth of cells in semi-soft agar and in vivo assays using growth innude mice as indicative of the transformed phenotype. Additional methodsto determine the presence and expression levels of foreign DNA are alsowell-known, such as RT-PCR and Northern blots. Determination of themolecular size of genetic material containing probed sequences with andwithout exposure to specific endonucleases may be used to determinewhether foreign DNA has been integrated into the host cell genome.

[0050] The following examples illustrate, but not limit, the presentinvention.

EXAMPLE 1 Primary Culture of Pituitary Cells

[0051] A. Primary Culture of Bovine Pituitary Cells.

[0052] Fresh bovine pituitary glands were received from G&C Packing Co.(Colorado Springs, Colo.) within 6 hours of extraction. These werehandled aseptically with sterile solutions throughout as follows: 1)Extraneous tissue was removed and the glands were quickly immersed in70% ethanol and rinsed with Krebs-Ringer buffer solution containing 0.5%BSA, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical Co.,St. Louis, Mo.). 2) The glands were mechanically dissociated by mincingwith a scalpel and sieving through a metal screen. 3) The homogenate wasthen treated with Krebs-Ringer buffer containing 0.2% trypsin, 1 mg/mlhyaluronidase and 1 mg/ml collagenase (Sigma Chemical Co., St. Louis,Mo.) for 60 minutes at 37° C. with agitation. 4) Enzymes were washedfrom dispersed cells by suspension in Krebs-Ringer buffer andcentrifugation (10 minutes at 48×g) of pituitary cells. 5) Pelletedcells were resuspended in minimum essential medium (MEM) containing 5%FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% mycostatin. 6)Cells were plated in 6 well culture plates (Nalge Nunc International,Rochester, N.Y.) using 1 pituitary gland per 4 wells; about 1×10⁵ cellsper ml and maintained in humidified air, supplemented with 5% CO₂ at 37°C. Plated cells were visualized with an inverted, phase contrastmicroscope.

[0053] B. Primary Culture of Human Pituitary Cells.

[0054] Fetal human pituitary glands were obtained from Anatomical GiftFoundation (White Oak, Ga.) or Advanced Bioscience Resources, (Alameda,Calif.). Pituitary glands were stored in approximately 20 mls of RPMIculture media at 4° C. and processed within 24 hours of dissection. Thegestational age ranged from 19 to 24 weeks. The following dispersionprocedure was performed using sterile materials and techniquethroughout. Tissue was rinsed in 30 mls modified Kreb's Ringer solution,supplemented with 5 g/L bovine serum albumin (BSA) and nonessentialamino acids. The tissue was then transferred to a glass microscopeslide, and finely minced using scapels. Minced tissue was suspended in 2to 2.25 mls modified Kreb's Ringer containing 1 mg/ml collagenase (SigmaChemical Co., St. Louis, Mo.). The mixture was incubated at 37° C. withrotation at 250 rpm for 60 minutes. The dispersed cells were washed in20 mls DMEM/F12 medium and centrifuged (5 minutes at 428×g). Thesupernatant was aspirated leaving 2-4 mls, and two additional washeswere performed using 10 mls DMEM/F12. Cells were counted using ahemacytometer (Fresney, I R, 1987). The total number of cells rangedfrom 100,000 to 2 million per gland, with greater cell numbers in olderpituitary glands. The mixture was centrifuged (5 minutes at 428×g). Thepellet was then resuspended in culture media, as described below, atapproximately 10⁵ cells/ml and plated into multi-well, styrene tissueculture plates, incubated at 37° C. in humidified air supplemented with5% CO₂.

[0055] Primary culture conditions necessary for the proliferation ofendocrine cells of the pituitary gland were investigated. The presentinvention requires cellular proliferation of target cells forintegration of foreign DNA into the host cell genome and induction ofimmortalization. The pituitary gland is composed of numerous cell typesincluding fibroblasts, follicular stellate cells, endocrine epithelialcells (gonadotropes, thyrotropes, somatotropes, lactotropes,melanotropes, corticotropes) and endothelial cells from blood vessels.Since the hormone-producing endocrine cells are one target of thepresent invention, we investigated methods to enrich the population ofendocrine cells. Fibroblast cells may be significantly depleted fromprimary cell cultures by differential adsorption to tissue cultureplates, a method known as “panning”. Primary cultures were panned byinitially plating the dispersed pituicytes in a single 35 mm tissueculture dish at about 5×10⁴ cells/ml. After 16-24 hour cells wereremoved from these cultures by repetitive pipetting of media(approximately 10 times). These cells were pelleted by centrifugation (5minutes at 428×g) and replated at 10⁵ cells/ml. Centrifugation at lowerg forces resulted in incomplete recovery of cells from the pellets.Hormone assay results and immunohistochemical staining showed that thehormone-producing cells were enriched in the less adherent cellpopulation while fibroblast cells were more strongly adsorbed. Whilethis panning method did not result in complete separation of fibroblastand endocrine cells, somatotropes and gonadotropes were significantlyenriched within the panned cultures.

[0056] Experiments were first performed to investigate the effect ofdifferent culture media on the growth of endocrine cell-enrichedcultures. We investigated a commercially available medium, Opti-MEM(Life Technologies, Rockville, Md.) with and without additional glucose(50 mg/ml and non-essential amino acids (Sigma Chemical Co., St. Louis,Mo.). We also tested a serum-free defined media, Gc, that has been usedin the primary culture of human prostate epithelial cells (Hedlund andMiller, 1994). The modified Gc media tested here utilized either RPMI1640 or Medium 199 (Life Technologies, Rockville, Md.), 20 μg/ml bovineinsulin, 10 μg/ml human apo-transferrin, 25 nM sodium selenite, 0.5 mMsodium pyruvate, 2 mg/ml BSA (Cohn fraction V), 10 mM HEPES, 0.5 mg/mlfetal bovine fetuin, 50 nM hydrocortisone, 10⁻¹⁰ M Tri-iodothyronine(T3), 1 ng/ml human recombinant EGF (Intergen, Co., Purchase, N.Y.).Unless otherwise indicated, all reagents were purchased from SigmaChemical Co. (St. Louis, Mo.). Cell growth was determined by periodiccell counts using a hemacytomer of less adherent cells that were removedby multiple pipetting. The results are shown in FIG. 1. There was aninitial decrease in the total number of cells at five days with allmedia tested. Opti-MEM failed to support growth regardless of additionalglucose or amino acids (broken lines). RPMI-based Gc media supportedmaximal logarithmic growth apparent between days 5 and 15. The Gc mediaand additions resulted in approximately 5-fold increases in the totalcell population at 15 days (solid lines). The RPMI 1640-based Gc mediumwas chosen for additional study because of its ability to supportgrowth.

[0057] To monitor the viability and/or secretory capability of thehormone-producing endocrine cells, we measured secretion of pituitaryhormones from cultures of panned pituitary cells. The level of FSH, LH,prolactin and TSH in culture media was determined using an automatedchemiluminescent-based immunoanalyzer, ACS: 180^(tm) (ChironDiagnostics, Inc., Norwood, Mass.). Growth hormone was determined usingan immunoradiometric assay (Nichols Institute, San Juan Capistrano,Calif.). Culture media was harvested on Mondays and Fridays from growingcultures for several weeks. These cultures were maintained in 48-wellplates (Falcon/Becton Dickinson, Franklin Lakes, N.J.), with each wellcontaining 1 ml of media. The majority of the media were carefullyremoved to avoid uptake of cells and transferred to 12×75 mm tubes foranalysis. Cells were fed with an equal volume of fresh medium at thesame time points. Background levels of GH in various media wereinsignificant (<0.1 ng/ml). Each condition was tested in duplicate andgraphed values are the mean hormone concentration +/− standarddeviation.

[0058]FIG. 2 shows the secretion of GH by panned pituicyes over thecourse of 15 days in various media. Estradiol (10⁻¹⁰ M) was included inthe media since previous experiments indicated its stimulatory effect onGH secretion. Monolayer cultures of pituicytes showed continualincreases in GH secretion over 19 days, while confluence was reached at12 days. We also tested a three-dimensional spheriod culture technique(Hedlund, T E, et.al., 1999) using the same Gc medium with estradiol.While spheriod cultures have been used to promote differentiation ofmany primary cultures, there was no apparent effect on GH secretion.RPMI-based Gc media resulted in higher levels of GH secretion thanDMEM/F12 based-media with or without estradiol (See FIG. 2).

[0059] Similar types of experiments were done to optimize cell cultureconditions and components for secretion of GH, FSH, LH, and prolactinfrom primary cultured human pituitary cells. The results are shown inTable 1. The tissue culture conditions investigated included the basemedia for Gc, addition of non-essential amino acids (NEAA) to the media,panning in Gc media and spheriod 3D culture, as described above. We alsoinvestigated pituicyte growth on plastic coated with Matrigel(Collaborative Biomedical Products, Bedford, Mass.). However, theinability to manipulate these cultures for propagation, transfection andimmunostaining precluded its use. Also investigated were three hormonesfrom the steroid receptor superfamily including: hydrocortisone,17β-estradiol, and T3. While 10⁻¹⁰ M T3 was beneficial to the secretionof GH, LH and prolactin, it was strongly inhibitory to TSH releaseprobably because of its well-known inhibitory effect on TSH genetranscription in thyrotropes. TABLE 1 Optimum Conditions for PituitaryHormone Secretion from Primary Cultures GH FSH LH PRL GENERAL T.C.CONDITIONS Base medium for Gc RPMI RPMI DMEM/ RPMI F12 Nonessentialamino acids + + + NE Panning ++ ++ + NE Spheriod 3D Culture No − − Noadvantage advantage STERIOD HORMONE SUPERFAMILY Hydrocortisone 5 × 5 ×NE NE 10⁻⁸  M 10⁻⁸  M 17β-Estradiol 1 × NE NE 1 × 10⁻¹⁰ M 10⁻¹⁰ MT3(3,5,3′ triiodothyronine) 1 × NE 1 × 1 × 10⁻¹⁰ M 10⁻¹⁰ M 10⁻¹⁰ MPEPTIDE HORMONES & GROWTH FACTORS GnRH NE 10⁻¹⁰ M 10⁻⁷ to NE 10⁻¹⁰ MSimiliar effect FGF-basic 0.1 ng/ml NE NE NE

[0060] We also investigated the effects of hypothalmic releasinghormones and select growth factors on pituitary hormone secretion. Whilethere are reports of beneficial effects of platelet-derived growthfactor (AA & BB) and FGF-acidic & basic (Leon, S P, et.al., 1994;Ericson, J, et.al., 1998) on cultured pituitary cells, we havepreliminary evidence to suggest that 0.1 ng/ml FGF-basic potentiates GHrelease. All other conditions were found to produce no detectable effecton GH, FSH, LH and prolactin release. GnRH (gonadotropin releasinghormone) was also tested in the presence of cortisone and estrogen. BothFSH and LH secretion were significantly potentiated with the initialtreatment, while repeated application of GnRH was progressively lesseffective. A concentration of 10⁻¹⁰ M GnRH was best for FSH and LH waseffected by all concentrations tested. For this reason there appears tobe no long-term advantage for including GnRH in the medium formulation.Hence the data shown in Table I enabled us to create an optimal mediumfor maintaining the secretion of GH, FSH, prolactin by pituicytes inculture. LH secretion, however, was poorly maintained even withconditions found to be optimal for FSH. This may reflect instability ofthe secreted LH and/or the lack of necessary factors to maintain LHsecretion.

EXAMPLE 2 Expansion of Primary Cultured Pituitary Cells

[0061] A. Expansion of Bovine Pituitary Cultures.

[0062] Confluent cultures were incubated with 0.025 mg/ml trypsin withEDTA (Life Technologies, Inc., Rockville, Md.), centrifuged (10 minutesat 47×g) and subcultured at 1×10⁵ cells/ml in small T-flasks (T-25).After 3 to 5 passages, these cultures exhibited signs of senescenceincluding the appearance of large vacuolated, multi-nucleated cells anda reduced growth rate.

[0063] B. Expansion of Human Pituitary Cultures.

[0064] Confluent cultures were exposured to low levels of trypsin(0.0025 mg/ml trypsin in 0.05 mM EDTA; Sigma Chemical Co., St. Louis,Mo.) for 30 seconds to 1 minute. This reaction was immediately stoppedby the adding an equal volume of 0.2 mg/ml soybean trypsin inhibitor(Sigma Chemical Co, St. Louis, Mo.). Cell viability was diminished byfailure to inhibit trypsin. Cells were washed once by centrifugation (5minutes at 428×g) and were subcultured at 10⁵ cells/ml. These culturescontinued to grow at similar rates through 3 passages. By the fifthpassage, little further growth was observed even with prolonged culture.Hormone secretion was found to decrease with sequential passaging.Growth hormone secretion diminished by 50%, 85% and 100% in passages 2,3 and 4 respectively. Secretion of FSH and prolactin diminishedsimilarly. Hence, using these methods, senescence of primary culturedhuman pituitary cells occurs near the 5^(th) passage.

[0065] Flow cytometric immunofluorescence was used to determine thepercentage of first passage cells expressing growth hormone after 19days in primary culture. Cells were prepared by incubating in achelating buffer (135 mM NaCl;, 5 mM KCl, 20 mM Hepes, 1.5 mM EDTA, pH7.4) and repeatedly pipetted to detach the cells from culture plates.Large aggregates of cells were allowed to settle out for 1 minute. Thesuspended cells were fixed in an equal volume of Zamboni fixative(Hatfield, J M and Hymer, W C, 1985) for 20 minutes at room temperatureand then washed three times in 10 mls PBS by centrifugation (5 minutesat 428×g).

[0066] The final pellet was resuspended in 1 ml PBS, 5% goat serum and0.1% sodium azide. Incubation in serum continued for 10 minutes at roomtemperature to block nonspecific binding. After centrifugation, thecells were incubated with either of two primary antibodies at 2 μg/ml: amouse monoclonal antibody to GH (Biogenesis, Inc., Catalog No.4750-0280) or a nonspecific mouse IgG₁ (Dako Corp., Catalog No. X-0931).These mixtures were agitated every 10 minutes for the first half hourand then incubated overnight at 4° C. The cells were then washed threetimes with PBS containing 1% BSA and 0.1% azide.

[0067] The cells were reacted with the secondary antibody,FITC-conjugated goat anti-mouse Ig (Dako Corp., Catalog No. F-0479) at50 μg/ml. The cells were incubated at room temperature for 30 minutesand washed twice with PBS. The final pellet was resuspended in 500 μlPBS, 1% BSA, 0.1% sodium azide and stored at 4° C. in the dark untilflow cytometric analysis.

[0068] The cells were analyzed on a Mo Flow (Cytomation, Inc., FortCollins, Colo.) flow cytometer. Forward and side-scatter profiles wereused to gate intact, single cells. The fluorescence of nonspecific andgrowth hormone-stained cells was quantitated and compared. The percentpositive cells were monitored, setting the nonspecific at 2.9% andsubtracting it from the final number. This analysis indicated that 34%of the pituicytes were positive for growth hormone. Furthermore, themean fluorescence of the GH-stained cells was 11-fold brighter than thenonspecifically stained population. These results indicate that at leastone third of our expanded pituitary cultures express growth hormoneafter 19 days in culture. This percentage of somatotropes is verysimilar to that of the intact, adult pituitary gland.

[0069] The percentage of proliferating cells in the human pituitarycultures was also quantitated using flow cytometry. We utilized livecells from the suspension described above for GH staining. Approximately5×10⁴ cells were pelleted and resuspended in 500 μl of propidium-iodidesolution containing saponin and RNAse (Hedlund, et.al., 1998). Theproliferating cell population was quantitated by adding together theS-phase and G₂/M peaks. Approximately 17% of the cells wereproliferating at this time point. This relatively high mitotic rate wascritical to maximize transfection efficiency for subsequentimmortalization of primary cultured human pituitary cells.

EXAMPLE 3 Transfection of Cultured Pituitary Cells

[0070] A. Transfection of Bovine Pituitary Cells.

[0071] Transfection efficiency in cultured pituitary cells wasdetermined using the pcDNA3 plasmid containing the β-galactosidase gene(InVitrogen, Inc., San Diego, Calif.) and calorimetric detection ofgalactosidase activity in cultured cells. Liposomes containing cationiclipids (e.g., Lipofectin®; Life Technologies, Inc., Rockville, Md.) werefound to be effective. FIG. 3 shows the results of optimization of theDNA/lipid ratios at two different plating densities. These data showthat 1 to 2 μg DNA per 4 to 6 μl Lipofectin® result in transfectionefficiencies slightly greater than 1% when the cells are plated at 3×10⁴cells/ml. Liposome-based transfection using cationic liposomes such asLipofectin® results in successful transfection of primary culturedbovine pituitary cells. The third pass culture of primary bovinepituitary cells were transfected at 60 to 70% confluency in the wells ofa 24 well plate. The transfection volumes of each well were 0.4 ml ofDMEM/F12 medium, which contained 0.5 μg of pSV3neo plasmid and 1.5 μl ofLipofectin®. The transfection solution was replaced 16 hours later withcomplete growth medium which is replaced every 2 to 3 days.

[0072] B. Transfection of Human Pituitary Cells.

[0073] Dispersed and panned cells human pituitary cells were plated at200,000 to 300,000 cells per ml in 6-well plates (Falcon/BectonDickinson, Franklin Lakes, N.J.) and grown until 30-50% confluent.Transfection was performed using cationic lipids to deliver plasmidscontaining either the SV40 large T antigen or the v-myc oncogene intoprimary-cultured pituitary cells. Plasmids without oncogenes were usedas controls for transfection. Wells transfected with control plasmidslightly outlived primary cell cultures due to the presence of the genecoding for G418 resistance but died after 2 to 3 weeks or a singlepassage of the cells. Using Lipfectin®, we transfected panned pituitarycells with either pSV3neo (Southern, P J and Berg, P J, 1982) orpLXSN-v-myc, constructed as described below. The concentration of DNAwas kept constant (typically 1-2 μg) in each well while the amount ofcationic lipid increased from 0-20 μl. The plasmid (1-2 μg) was mixedwith 100 μl of Gc medium and incubated for 45 minutes at roomtemperature. Increasing amounts of Lipofectin® (2-20 μl) were also mixedwith 100 μl of medium and incubated for 45 minutes at room temperature.The two solutions were then combined, mixed gently and incubated for 30minutes at room temperature. The panned cells were then washed with Gcmedium and 0.8 ml of fresh medium was added to each well. For eachtransfection, we then added 0.2 ml of the Lipofectin®/DNA mixture usinggentle swirling of the plates to distribute the complexes. Thetransfection mixtures were allowed to incubate at 37° C. for 8 to 12hours before replacing the medium with 2 ml of fresh Gc medium. After 48hrs, medium was replaced with Gc medium containing 0.5% FBS and 150μg/ml G418. The formation of colonies in the presence of G418 wasmonitored for a varying number of weeks. We found that 150 μg/ml G418was sufficient to kill all primary pituitary cultures tested.

EXAMPLE 4 Plasmids Used to Immortalize Cultured Pituitary Cells

[0074] A. pSV3neo Plasmid.

[0075] The pSV3neo plasmid was obtained from the American Type CultureCollection (ATCC), Rockville, Md. This plasmid was prepared from E. coli(HB101) cell lysates. Plasmid DNA was purified using standard minipreptechniques well-known in the art.

[0076] B. Cloning the v-myc Gene into the Retroviral Vectors, pLXSN andpLNCX2.

[0077] The plasmid, pSVv-myc, containing a fusion of the gag and v-mycgenes (Land, et.al., 1983; provided by Dr. Robert Weinberg, WhiteheadInstitute of Biomedical Research, Cambridge, Mass.) was amplified by PCRto obtain the v-myc gene sequence for cloning into two retroviralvectors, pLXSN which contains a weak promoter in the 5′ LTR region andpLNCX2 (See FIG. 4) which contains a constitutive promoter, CMV(Clonetech, Inc., Palo Alto, Calif.). Having both a weak promoter and astrong promoter to drive production of v-myc allows for two differentlevels of oncogenic protein that may have different effects in theimmortalization of pituitary cells.

[0078] The cloning steps are shown below for both pLXSN and pLNCX2derivatives. The same primer set was used for both PCR amplification ofv-myc and cloning into pLXSN. The 5′ primer contains an EcoRI site(underlined) and a start codon (residues 16-18) as follows:5′-CGAGCGGAATTCGCCATGGTGCAC GGCCAGGCAGC-3′ (SEQ.ID.NO.1). The 3′ primercontains a BamHI site (underlined) and a stop codon (residues 13-15): 5′CGAGCGGGATCCCTATGCACGAGAGTTCCTTAGCTGCTC-3′ (SEQ.ID.NO.2). Theseoligonucleotides at 1 μM were used for PCR in the presence of 4 units ofTaq DNA polymerase (Promega, Cat. No. M1665), 10 mM Tris-HCL(pH 9), 50mM KCl, 0.1% Trition X-100, 1.5 mM MgCl₂, 200 μM dNTPs and 10% DMSO toobtain the v-myc gene sequence for cloning. Thirty cycles of PCR wereperformed as follows: 1 minute at 94° C. for denaturation, 1 minute at66° C. for annealing, and 3 minutes at 72° C. for extension. Theresulting product and the pLXSN vector were then cut with an excess ofEcoRI and BamHI overnight at 37° C. The DNA digests were purified usinga PCR purification kit (Qiagen, Inc., Cat No. 28106). The cut vector andPCR product (about 1300 bp) were mixed at a 1:3 molar ratio and ligatedovernight with T4 DNA Ligase (Promega, Inc., Cat No. M1804) at 16° C.The ligation mixtures were then diluted 1 to 10 and for transformationof competent DH5α cells (Life Technologies, Inc., Cat. No. 18265-017).Transfornants were selected on LB agar containing 100 μg/ml ampicillin.Individual clones were expanded and plasmid DNA was isolated usingwell-known miniprep techniques. Diagnostic restriction digests wereperformed using EcoRI, BamHI and SalI. Cleavage with SalI in combinationwith the original enzymes used for cloning, EcoRI and BamHI, yielded 600and 700 bp fragments that were diagnostic for the presence of v-mycsince the Sall site is not present in the pLXSN vector. One clone wasselected and sequenced. It showed complete homology to the reported genesequence of v-myc (Atitalo, K, et. al., 1983). The resultant vector,pLXSN-v-myc was used to transfect primary cultured pituitary cells usingthe methods described in the previous section.

[0079] Primers for PCR and cloning of the v-myc gene into the pLNCX2vector differ only in the restriction sites used for cloning. The 5′primer contains a HindIII site (underlined) and a start codon (residues16-18) as follows: 5′-CGAGCGAAGCTTGCCATGGTGCACGGCAGGCAGC-3′(SEQ.ID.NO.3). The 3′ primer contains a StuI site (underlined) and astop condon (residues 13-15): 5′-CGAGCGAGGCCTCTATGCACGAGAGTTCCTTAGCTGCTC-3′ (SEQ.ID.NO.4). The PCR and cloning steps are the same forcloning into the pLNCX2 vector.

EXAMPLE 5 Selection of Transfectants

[0080] A. Bovine Cells.

[0081] Since the pSV3neo plasmid contains the neomycin resistance gene,selective pressure was applied to transfected bovine pituitary cellsusing 300 μg/ml G418, . Bovine pituitary cells transfected with theplasmid bearing the SV40 large T antigen (pSV3neo) outlive control cellstransfected with a naive plasmid, which rarely survive beyond the sixthor seventh passage in culture. The pSV3neo transfected cells have beenroutinely cultured, stored cryogenically, and have been passaged up to48 times for over a year without noticeable variations in growthpatterns.

[0082] B. Human Cells.

[0083] Primary cultured human pituitary cells transfected with pSV3neowere incubated for an additional 48 to 60 hours in Gc medium with 10⁻¹⁰M estradiol and nonessential amino acids before G418 selection. G418(Sigma Chemical Co., St. Louis, Mo.) was then added at 150 μg/ml forfive days and then increased to 250 μg/ml for subsequent culture. Thisfinal dose was sufficient to kill 100% of non-transfected cells. Mediumwas replaced every 3 to 4 days and the formation of individual colonieswas monitored visually. When colonies occupied at least half of thewell, we subcultured the cells into new 6-well plates in Gc mediasupplemented with nonessential amino acids and 0.5% fetal bovine serumand 250 μg/ml G418. The cultures were then expanded, and frozen inliquid nitrogen in medium containing 10% DMSO. Cloning was performed bydiluting cells in Gc medium plus 0.5% FBS and 250 μg/ml G418 to aboutone cell per well in 96-well plates. Wells with more than one cell thatformed colonies were not used. Clonal cell lines were then propagated,characterized as described below and frozen as above.

EXAMPLE 6 Characterization of Immortalized Cells

[0084] A. Bovine Pituitary Cell Lines.

[0085] Initial characterization of bovine pituitary cells transfectedwith the pSV3neo plasmid showed that these cells were immortalized.Operational immortalization was established by the survival of thevarious lines of immortalized bovine cells up to 48 passages. (Thepassage number of the various lines of immortalized bovine pituitarycells is shown below in Table 2). Non-transformed cells undergosenescence after 6 to 7 passages. Although there were minor variationsin growth rates during expansion of these cultures, there was noapparent decrease in growth rate.

[0086] Also, the presence of the pSV3neo plasmid in these cell lines wasdemonstrated by a) survival of clones in doses of G418 (up to 4 mg/ml)lethal to wild type, non-transfected cells, and b) detection of the mRNAof the large T antigen and the neomycin resistance gene by RT-PCR of thepSV3 neo transfected cells. These results indicate the presence of thelarge T antigen sequence within the transfectants and suggest thatimmortalization is a consequence of the expression of the large Tantigen.

[0087] We then tested these immortalized cells by two well-known methodsto analyze malignant transformation of cells. First, cell growth in softagar was determined (Freshney, 1987). A completely transformed cell linehas reduced requirements for cell attachment and can form colonies insemi-soft agar without attachment to solid surfaces. Absence of growthin soft agar is one established criterion of a non-transformed cellline. TABLE 2 Cell lines tested for growth in semi-soft agar. CELL LINEPASSAGE NUMBER bGH C1 26 bGH C1+ 26 bGH C5 26 bGH C6+ 26 bGH C3-1 37 Tag2 48 Tag 2+ 48 MCF-7 w.t. (Positive control) — bp716D02 w.t. (Negativecontrol)  2

[0088] Table 2 shows cells tested for growth in soft agar (RA Freshney,1987). The first seven cell lines listed were transfected by the pSV3neoplasmid and were tested at the passage listed. The (+) symbol after someof the cell lines indicates that the cells were cultured in 500 μg/mlG418. Wild type (w.t.) bovine pituitary cells (bp716D02) were used as anegative control and the transformed MCF-7 cells were the positivecontrol.

[0089] The cells were diluted into soft agar at different concentrationsin a six well plate as indicated in Table 3. The cells were also platedin growth medium without agar to control for viability. TABLE 3 Well 1:1000 cells/well in soft agar Well 2:  333 cells/well in soft agar Well3:  111 cells/well in soft agar Well 4:  38 cells/well in soft agar Well5 & 6: 6000 cells/well in DMEM/F12 5% FBS

[0090] Cultures were maintained at 37° C. for 15 days in a humidifiedincubator with 5% CO₂. Colonies were visualized and counted only if thecolony was not attached to the bottom of the plate, was spheroid andmoved within the soft agar. The results are shown in Table 4. TABLE 4Growth of various cell lines in semisoft agar. Cell Line % of controlavg % colonies/cells plated MCF-7 w.t. 100    18.2  (Positive control)bGH C5 7.7 1.4 bGH C6+ 0.7  0.13 bGH C1+ 0.3  0.05 Tag 2 0.2  0.03 bGHC1 0   0   bGH C3-1 0   0   Tag 2+ 0   0   bp716D02 w.t. 0   0  (Negative control)

[0091] Colony formation is presented as a percentage of the number ofcells plated as well as a percent of the MCF-7 positive control. Four ofthe bovine cell lines failed to form detectable colonies, while othersshowed very low growth in comparison with the MCF-7 positive control.Line C5 showed 7.7% of the growth of the MCF-7 cells.

[0092] Additional studies were performed to determine if theimmortalized bovine pituitary cell lines would produce tumors in athymicmice. Ten million cells were subcutaneously injected into each of fiveBalbC Nu Nu mice and tumor size (mm³) was measured weekly for up to 90days. We tested MCF-7 cells (ATCC, Rockville, Md.) as a positive controland the immortalized bovine pituitary cell lines, C3-1 and C1+. Miceinjected with MCF-7 cells showed progressive tumor growth until reachinga mass which was fatal, about 600 mm³ or less (FIG. 6A). (Micerepresented by ⋄ or Δ died at 26 and 33 days respectively.) Thisindicates that the transformed MCF-7 cells rapidly proliferate inathymic mice. However, the results obtained with the bovine cell linesC1+ (FIG. 5B) and C3-1 (FIG. 5C) showed an absence of progressive tumorgrowth with most mice showing no detectable tumors. Three out of 10 micedid exhibit measurable tumors at about 30 days post implant, but thesetumors were only 10% or less of the size of the MCF-7 tumors andregressed to zero (2 mice) or minimal (1 mouse) volume at 90 days postimplantation. The Y axis scales on FIGS. 5A, B and C are different. Theresults of the soft agar and nude mouse studies indicate that theimmortalized bovine pituitary cells exhibit two characteristics ofnon-transformed cells: absence of growth in soft agar and inability toform tumors in athymic mice.

[0093] The immortalized bovine pituitary cell lines were thencharacterized by immunohisto-chemical staining specific for particulartypes of cells. Bovine cultures were grown in chamber slides (NalgeneNunc, Inc., Naperville, Ill.) until approximately 50% confluent and wererinsed 3 times with PBS. For the pituitary hormone stains, cells werefixed with Zamboni fixative (Hatfield, J M and Hymer, W C, 1985) for 20minutes at room temperature. For staining of intermediate filaments,cells were fixed in 70% methanol/30% acetone at −20° C. for 10 seconds.After fixation, cells were rinsed twice in PBS. Nonspecific binding wasblocked by incubating in PBS with 5% goat serum and 0.1% sodium azidefor 20 minutes in a humidified incubator at 37° C. After rinsing threetimes in PBS, each chamber received approximately 2 μg/ml of theappropriate primary antibody: 1) rabbit anti-goat polyclonal Ig, anonspecific control (Sigma Chemical Co., Catalog No. B-7014), 2) rabbitanti-bovine growth hormone (Biogenesis, Inc., Catalog No 4750-0959), 3)rabbit anti-bovine FSH (Biogenesis, Inc., Catalog No. A558/R4H), 4)Mouse nonspecific IgG₁ (Dako Corp., Catalog No. X-0931), 5) mouseanti-vimentin monoclonal IgG₁, (Sigma Chemical Co, Clone No. V-9), 6)mouse anti-cytokeratin pan AE1/AE3 monoclonals (Dako Corp., Catalog No.M-3515). Cells were incubated for 30 minutes at 37° C. with the primaryantibodies and were washed three times in PBS.

[0094] The secondary antibodies were applied at a 1:500 dilution in PBS,1% BSA and included: peroxidase-conjugated goat anti-mouse IgG (JacksonLaboratories, Catalog No. 115-035-062) or peroxidase-conjugated donkeyanti-rabbit IgG (Jackson Laboratories, Catalog No. 711-035-152). Cellswere incubated for 30 minutes at 37° C. and washed four times in PBSwith 1% BSA. Cells were then reacted in 667 mg/ml diaminobenzidine(Sigma Chemical Co., St. Louis, Mo.) with a 1:1000 dilution of 30%hydrogen peroxide. This reaction was stopped after 10-20 minutes byrinsing four times in deionized water. Slides were counterstained withhematoxylin and preserved using an aqueous permanent mounting medium,Ultramount (Dako Corp., Carpinteria, Calif.)

[0095] The results of the immunohistochemical staining showed that allbovine cultures were strongly positive for vimentin, a common feature ofimmortalized and transformed cells, as well as normal fibroblasts. Whilethe original bovine primary cultures showed strong staining for theepithelial cytokeratins in approximately 2% of the cells, more uniformstaining was detected in clones C3, C6 and Tag2. We were unable todetect GH or FSH by immunostaining. However, the limited sensitivity ofthe immunostaining methods may preclude our ability to detect low levelsof expression.

[0096] Additional experiments were performed using highly sensitiveimmunological methods to detect bovine growth hormone within live,individual cells in the immortalized cultures. Bovine GH-specificmonoclonal antibodies were obtained from Biogenesis, Inc. (Catalog No.4750-0939) and OEM Concepts (Toms River, N.J.). The specific sensitivityand specificity of these antibodies was confirmed as follows: Animmunoblot assay procedure showed a visibly linear response tocommercial bovine GH (Biogenesis, Inc., Sandown, N.H.) from 10 to 100ng/100 uL. Western blots of commercial preparations of purified bovineGH and bovine pituitary extracts showed reactivity of a single band atthe molecular size of bovine GH. A cellular blotting method was used tomonitor bovine GH secretion from pituitary cells grown in culture, asdescribed by Gibson-D'Ambrosio, et al. (1995). By localizing bovine GHwithin cultures, it was possible to obtain enriched cultures of bovinesomatotropes prior to transfection with pSV3neo. Subsequent generationsof these transfected clones showed enrichment of the proportion of cellssecreting bovine GH. These cell lines are indicated in Table 2 with thedesignation bGH, e.g., bGH C5. However, the enriched bovine GH culturesalso contained cells that do not appear to secrete bovine GH, indicatingthat the surviving cells are heterogeneous.

[0097] Immunoblots showed a positive signal from FBS-containing mediaprobably due to bovine GH in FBS since non-FBS containing media wasnegative. To test for secretion of bovine GH from immortalized bovinecell line, cultures of passage 2-10 were grown to confluence inFBS-containing media, washed three times with PBS and incubated for 3hours in media without FBS. Duplicate wells contained 10 μM bovinegrowth hormone releasing factor (GHRF). The results showed that theconditioned media contained detectable levels of bovine GH and theamount secreted appeared to be potentiated by exposure of the culturesto GHRF. These results suggest that cultures of immortalized bovinepituitary cells secrete bovine GH into the culture medium.

[0098] Further analysis was performed to determine the presence ofbovine GH within these cultures. A Western blot procedure according tothe method of Fernandez and Kopchick (1990) was performed using apolyclonal antibody to bovine GH (Biogenesis, Inc., Catalog No.4750-0959). The results showed a band at the approximate molecularweight of bovine GH in the extracts of cell lines C5, C6, Tag1 and Tag2as well as early passage wild-type cells. However, several otherresolvable bands were also immunoreactive, possibly due tocross-reaction of the primary polyclonal antibody. RT-PCR was used todetermine if the GH mRNA could be detected within the immortalizedbovine pituitary cells. Using primers to exon 1 and exon 4, we were ableto detect amplification of a correctly-sized DNA in cell lines C1, C5and C6 at the sixth passage. This signal was also detected in lowpassage primary cultures and digestion of the RT-PCR product with Smalyielded two bands of the predicted size.

[0099] In summary, the characterization of bovine pituitary cellsimmortalized by transfection with the pSV3neo plasmid shows that theresulting cell lines were immortal and contained the pSV3neo plasmidwhich probably induced immortalization through expression of the large Tantigen. The immortalized bovine pituitary cells appear non-transformedby two commonly used methods to assess cellular transformation, growthin semi-soft agar and athymic mice. By immunohisto-chemical staining,the co-expression of the cytokeratins and vimentin in several cell linessuggests that epithelial cells have been immortalized by transfectionwith the pSV3neo plasmid. The RT-PCR results indicate that thesecultures express the mRNA for large T antigen and bovine growth hormone.Additional immunological analyses by different monoclonal and polyclonalantibodies show the presence of bovine growth hormone and preliminaryresults suggest that it is secreted. Taken together the results stronglysuggest the presence of immortalized bovine somatotropes within thesecultures.

[0100] B. Human Pituitary Cell Lines.

[0101] Three different cell lines were isolated from transfectionexperiments described above. These are shown below with passage number:TABLE 5 Cell lines tested for growth in semi-soft agar. CELL LINEPASSAGE NUMBER Human LTA5/pSV3neo 18 Human LTA6/pSV3neo 24 HumanLTA8/pSV3neo 21

[0102] Since these cell lines have been passaged well beyond the pointwhere primary human pituitary cultures die (pass 4-5), this is evidencefor an immortalized state for each cell line. We initially characterizedthese cells by determining the expression of the large T antigen mRNAwithin these immortalized clones. We isolated total RNA from LTA5, LTA6and LTA8 cell lines by well-known methods in the art. This RNA was thenused to perform RT-PCR using primers specific for the 5′ and 3′ ends ofthe entire large T antigen sequence. The primers were: the 5′ primer,5′-GCAGCTAATGGACCTTCTAGGTC-3′ (SEQ.ID.NO.5) and the 3′ primer,5′-GTCAGCAGTAGCC TCATCATCAC-3′ (SEQ.ID.NO.6). Reverse transcription wasperformed using random hexamers under the following conditions: thereaction mixture contained 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl, 10 mM DTT, 1 mM dNTPs, 1 unit/μl RNAsin (Promega, Catalog No.N2111), 2.5 μM random hexamers (Promega, Catalog No. C1181), 2 units/μlMoloney Murine Leukemia Virus reverse transcriptase (Promega, CatalogNo. M1701, and 12.5 ng/μl of RNA from the immortalized pituitary celllines were incubated at 22° C. for 15 minute then moved to 42° C. for 45minutes. The samples were then heated for 5 minutes at 95° C. and thenstored on 5 μl volume was then removed from the reverse transcriptionreaction and mixed in a 25 μl PCR reaction containing 1 μM 5′ and 3′large T antigen primers, 4 units of Taq DNA polymerase (Promega, CatalogNo. M1665), 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Trition X-100, and1.5 mM MgCl₂. No additional dNTPs were added to the reaction with thefinal contribution from adding 5 μl of the RT reaction being 0.2 mM. Thereactions were incubated initially for 5 minutes at 95° C. and thenincubated for thirty cycles of 95° C. for lminute, 64° C. for 1 minute,and 72° C. for 1 minute. The resulting RT-PCR products were thenanalyzed by agarose gel electrophoresis.

[0103] The products from all three reactions contained three dominantbands for large T antigen but varied in predominance depending on theRNA used in the RT-PCR. For LTA5, the predominant product was thepredicted 670 bp band with much less intense bands at 500 bp and 320 bp.For LTA 6 and LTA8, the 320 bp band was much more intense than thelarger bands. This result suggested that different forms of large Tantigen were present in each cell line possibly due to alternatesplicing (Eul, J, et.al., 1996). Using the products generated in theRT-PCR reactions, restriction enzyme analysis was used to map the largeT antigen sequence present in each cell line. Restriction enzymes werechosen that span the entire length of the intact large T antigensequence. These were: SspI at 44 bp, Rsa I at 142 and 253 bp, NdeI at290 bp, TaqI at 390 bp, AluI at 486 bp, HinfI at 536 and 560 bp, andBfaI at 646 bp. The 670 bp band from the LTA5 RT-PCR product was cut byall the restriction enzymes except BfaI at 646 bp. Therefore, the LTA5contained large T antigen with coding sequences at least through 560 bp.The resulting restriction products from LTA6 and LTA8 showed that theyboth contain smaller forms of the large T antigen sequence. NdeI at 290bp, TaqI at 390 bp, or AluI at 486 bp did not cut the smaller species ofT antigen. Both of the smaller bands were cut by HinfI however. Theseresults suggest that LTA6 and LTA8 contain primarily a form of large Tantigen that were deleted for the middle portion of the sequence beyond253 bp through ˜500 bp. This is not surprising since the large T antigencan undergo alternate splicing of exons 1 and 2 to yield different formsof the antigen but still immortalize cells with only the amino terminusrequired (Asselin, C and Bastin, M, 1985).

[0104] These cell lines were also characterized with respect to theirgrowth in soft agar as described previously in the characterization ofthe immortalized bovine pituitary cells. Cell lines transfected by thepSV3neo plasmid and surviving senescence were compared to primary humanpituitary cells and tumorigenic wild type MCF-7 cells.

[0105] The cells were diluted in soft agar as shown in Table 6. Cellswere also plated in Gc medium+NEAA to control for viability. TABLE 6Well 1: 1000 cells/well in soft agar Well 2:  333 cells/well in softagar Well 3:  111 cells/well in soft agar Well 4:  38 cells/well in softagar Well 5 & 6: 6000 cells/well of human primary pituitary    culturehp991112, pass 2.

[0106] Table 7 summarizes the results at the highest concentration ofcells plated and compares them to the results obtained with MCF-7 cells.TABLE 7 Growth of Human Pituitary Cell Lines in semisoft agar. Cell Line% of control avg % colonies/cells plated MCF-7 w.t. (Positive control)100 72 Human LTA5(18)/pSV3neo 0 0 Human LTA6(24)/pSV3neo 0.035 0.025Human LTA8(21)/pSV3neo 12.6 9.1 hp991112(2) 0 0

[0107] The pSV3neo transfected cell lines LTA5 and LTA6 are probablynon-transformed cell types by their lack of growth in semisoft agar. TheLTA8 cell line showed a slight ability to grow in semisoft agar but mayalso be immortalized.

[0108] Human pituitary cells were prepared for immunohistochemicalstaining by the same methods that were previously described for bovinecells. Since the vimentin and cytokeratin antibodies were notspecies-specific, the same monoclonal antibodies were utilized for humanand bovine staining. The following human-specific antibodies were alsoused: 1) Mouse anti-human E-cadherin monoclonal IgG₁ (Clone HECD-1,Zymed Laboratories, San Francisco, Calif.), 2) mouse anti-GH monoclonalIgG₁ (Biogenesis, Inc., Catalog No., 4750-0280), 3) mouse anti-human FSHalpha subunit IgG₁ (Clone MAB111, AbProbe International, Portland, Me.).

[0109] The results are summarized in Table 8 and include staining ofboth primary cultures and immortalized cell lines. Strong staining forGH, FSH and cytokeratins was apparent within heterogeneous colonics ofthe primary cultured pituitary cells. Vimentin staining was rare in theprimary cultures, suggesting minimal contamination by fibroblasts. Thisconfirms that the panning method is effective in removal of fibroblastsfrom these cultures. Although GH and FSH could not be detected in thepSV3neo transfectants, two of the clones expressed markers of anepithelial phenotype. LTA8 expressed both E-cadherin and cytokeratin andLTA6 expressed only the cytokeratins. LTA5 expressed neither epithelialmarker. As with the bovine cell lines, the expression of vimentin wasubiquitous after immortalization. However, the expression of epithelialmarkers suggests that we have immortalized human epithelial cells, whichmay include endocrine cells, follicular stellate cells or endothelialcells. The immortalized cell lines were further characterized by RT-PCRanalysis of the messenger RNA of common pituitary hormones. TABLE 8Immunostaining of Human Fetal Pituitary Cultures E- Cyto- Cells Cadherinkeratins Vimentin GH FSH HP990821 (primary) + + + + ? HP990902(primary) + + + + − HP990923 (primary) + + + + + LTA5 (immortalized) −− + − − LTA6 (immortalized) − + + − − LTA8 (immortalized) + + + − −

[0110] Total RNA was isolated from LTA5, LTA6, and LTA8 pituitary celllines using methods well-documented in the art. These RNAs were thenanalyzed by RT-PCR for the presence of messages for the pituitaryhormone α-subunit, growth hormone, and prolactin. The α-subunit iscommon to FSH, TSH and LH. Cells dispersed for primary pituitarycultures were used as controls for the presence of all three mRNAs.RT-PCR was performed as described above with specific primers toα-subunit, growth hormone, and prolactin. The following primers wereused for the PCR portion of the analysis: α-subunit 5′ primer,5′-GCAGCTATCTTTCTGGTCACATTG-3′ (SEQ.ID.NO.7) and 3′ primer,5′-GTGGCACGCCGTGTG-3′(SEQ.ID.NO.8); growth hormone 5′ primer, 5′-CTGGCTTCAA GAGGGCAG-3′(SEQ.ID.NO.9) and 3′ primer,5′-CGTAGTTCTTGAGCAGTGCGT-3′(SEQ.ID.NO.10). The reactions were firstincubated for 5 minutes at 95° C. and then incubated for forty cycles of95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1 minute. Theresulting RT-PCR products were then analyzed by agarose gelelectrophoresis (see FIG. 6A). The RT-PCR products obtained from primarypituitary RNA contained a 300 bp band for α-subunit, a 503 bp band forgrowth hormone, and a 557 bp band for prolactin. The α-subunit productwas fully digested with XbaI to give 151 and 149 bp products and withHinfI to yield a 217 and 83 bp products. Growth hormone was digestedwith BanII to yield 215 and 288 bp products and prolactin cleaved byPvull generated 196 and 361 bp products. RT-PCR of LTA5, LTA6, and LTA8RNA gave no specific products corresponding to those found for growthhormone and prolactin from primary pituitary cells. There were specificproducts generated from RT-PCR using primers specific for α-subunit at˜300 bp (see FIG. 6B). The RT-PCR product for LTA8 appears to beslightly smaller than that generated from the primary pituitary cells.When the resulting products for all three cell lines were digested withXbaI and HinfI, all are cleaved by HinfI suggesting the sequences werefrom α-subunit. However, there was no digestion of the products withXbaI. Because the primary pituitary cells are of different origin thanthe cells used for immortalization, there may be a mutation present inthe pituitary used to generate the cell lines. Alternate splicing of thefirst intron is also known to result in restriction length polymorphisms(Fiddes, J C and Goodman, H M,1981). This may account for the differencein size observed between the LTA8 and primary pituitary cell RT-PCRproducts. Other nonspecific bands are also present as a consequence ofincreasing the number of PCR cycles from 30 to 40. These bands were alsopresent when RT-PCR was performed on primary pituitary cell RNA using 40PCR cycles.

[0111] In summary, human pituitary cells were immortalized bytransfection with the pSV3neo plasmid and these cells appear to benon-transformed by their lack of growth in soft agar. Some of thesecells appear to be epithelial by immunohistochemical staining forE-cadherin and cytokeratins. RT-PCR analysis showed a lack of the mRNAfor prolactin or growth hormone. However, the α-subunit mRNA wasapparently present, but this mRNA may be mutated at the site requiredfor digestion by XbaI. These cultures also did not secrete detectablelevels of FSH, LH or TSH, suggesting that large T antigen expression mayinterfere with the expression of human pituitary hormones.

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[0194] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare with the scope of the present invention and are only limited by thefollowing claims.

1. A method of immortalizing non-transformed cells comprising the stepsof: (a) obtaining a primary culture of cells; (b) transfecting saidprimary cells with a vector containing a foreign gene to producetransfected, immortalized cells that are not transformed; and (c)isolating said transfected cells from non-transfected cells to obtain aculture of immortalized cells, wherein said immortalized cells arepituitary cells, neurons, beta islet cells, glial cells, cornealepithelial cells or follicular stellate cells.
 2. The method of claim 1,wherein said primary cells are transfected by cationic liposomes,retroviruses or immunoliposomes.
 3. The method of claim 1, wherein saidprimary cells are transfected by immunoliposomes.
 4. The method of claim1, wherein said primary cells are hormone-producing cells.
 5. The methodof claim 1, wherein said primary cells are isolated with immobilizedantibody specific for cell surface proteins or hormone receptorsexpressed by said primary cells prior to transfection.
 6. The method ofclaim 1, wherein said primary cells are cultured with an extracellularmatrix protein.
 7. The method of claim 6, wherein said extracellularmatrix protein is fetuin, matrigel basement membrane matrix, ordisrupted MCF-7 cells.
 8. The method of claim 6, wherein saidextracellular matrix protein is fetuin.
 9. The method of claim 1,wherein said primary cells are pituitary cells.
 10. The method of claim9, wherein said pituitary cells are lactotropes, somatotropes,thyrotropes, gonadotropes, corticotropes or melanotropes.
 11. The methodof claim 1, wherein said primary cells are human cells.
 12. The methodof claim 1, further comprising culturing said primary cells with atleast one environmental factor to control proliferation ordifferentiation prior to or after transfection.
 13. The method of claim12, wherein said environmental factor is a growth factor or other factorcontrolling the expression of the foreign gene.
 14. The method of claim13, wherein said growth factor is activin, tri-iodothyronine, galinin,nerve growth factor, leukemia inhibitory factor, hepatocyte growthfactor, acidic or basic fibroblast growth factor, platelet-derivedgrowth factor, pituitary adenylate cyclase-activating polypeptide,vasopressin, retinoic acid, vasoactive intestinal polypeptide.
 15. Themethod of claim 1, wherein said vector is a retroviral expressionvector.
 16. The method of claim 15, wherein said retroviral expressionvector is pLNCX2 or pLXSN.
 17. The method of claim 16, wherein saidretrovial expression vector contains an inducible promoter.
 18. Themethod of claim 17, wherein said inducible promoter is derived from betagalactosidase, T7, mousemetallothionen, glucocorticoid or tetracyclineresponse elements..
 19. The method of claim 1, wherein said foreign geneis an establishment oncogene.
 20. The method of claim 19, wherein saidestablishment oncogene is a viral oncogene, a cellular proto-oncogene, atumor suppressor gene or a
 21. The method of claim 20, wherein saidviral oncogene is large T antigen of SV40, the Epstein-Barr virus, or E7gene of human papilloma virus.
 22. The method of claim 21, wherein saidviral oncogene is large T antigen of SV40.
 23. The method of claim 20,wherein said cellular proto-oncogene is gsp, gip², myc, fos or pituitarytransforming gene.
 24. The method of claim 20, wherein said cellularproto-oncogene is myc.
 25. The method of claim 1, further comprisingtransfecting said primary cells with an anti-apoptosis gene.
 26. Themethod of claim 25, wherein said anti-apoptosis gene is a Bcl2 gene. 27.The method of claim 1, wherein said immortalized cells remain viable atleast at a 5-fold greater passage number than senescence ofnon-immortalized primary cells under similar culture conditions.
 28. Animmortalized cell line produced by the method of claim
 1. 29. Theimmortalized cell line of claim 28, wherein said immortalized cell lineis a pituitary, neuronal, beta islet, glial, or corneal epithelial cellline.
 30. The immortalized cell line of claim 29, wherein saidimmortalized cell line is a pituitary cell line.
 31. The immortalizedcell line of claim 28, wherein said immortalized cell line is culturedin media free of other cell lines.
 32. The immortalized cell line ofclaim 30, wherein said immortalized cell line produces prolactin, growthhormone, luteinizing hormone, follicle-stimulating hormone,thyroid-stimulating hormone, adrenocorticotropic hormone ormelanocyte-stimulating hormone.